A multiscale, vertical-flow perfusion system with integrated porous microchambers for upgrading multicellular spheroid culture.

Spheroid formation assisted by microengineered chambers is a versatile approach for morphology-controlled three-dimensional (3D) cell cultivation with physiological relevance to human tissues. However, the limitation in diffusion-based oxygen/nutrient transport has been a critical issue for the densely packed cells in spheroids, preventing maximization of cellular functions and thus limiting their biomedical applications. Here, we have developed a multiscale microfluidic system for the perfusion culture of spheroids, in which porous microchambers, connected with microfluidic channels, were engineered. A newly developed process of centrifugation-assisted replica molding and salt-leaching enabled the formation of single micrometer-sized pores on the chamber surface and in the substrate. The porous configuration generates a vertical flow to directly supply the medium to the spheroids, while avoiding the formation of stagnant flow regions. We created seamlessly integrated, all PDMS/silicone-based microfluidic devices with an array of microchambers. Spheroids of human liver cells (HepG2 cells) were formed and cultured under vertical-flow perfusion, and the proliferation ability and liver cell-specific functions were compared with those of cells cultured in non-porous chambers with a horizontal flow. The presented system realizes both size-controlled formation of spheroids and direct medium supply, making it suitable as a precision cell culture platform for drug development, disease modelling, and regenerative medicine.

Expansion of Chinese hamster ovary cells via a loose cluster-assisted suspension culture using cell-sized gelatin microcarriers.

Technologies for efficiently expanding Chinese hamster ovary (CHO) cells, the primary host cells for antibody production, are of growing industrial importance. Various processes for the use of microcarriers in CHO suspension cultures have been developed, but there have been very few studies on cell-adhesive microcarriers that are similar in size to cells. In this study, we proposed a new approach to suspension cultures of CHO cells using cell-sized condensed and crosslinked gelatin microparticles (GMPs) as carriers. Unlike commercially available carriers with sizes typically greater than 100 µm, each cell can adhere to the surface of multiple particles and form loose clusters with voids. We prepared GMPs of different average diameters (27 and 48 µm) and investigated their effects on cell adhesion and cluster formation. In particular, small GMPs promoted cell proliferation and increased IgG4 production by the antibody-producing CHO cell line. The data obtained in this study suggest that cell-sized particles, rather than larger ones, enhance cell proliferation and function, providing useful insights for improving suspension-culture-based cell expansion and cell-based biologics production for a wide range of applications.

Process simplification and structure design of parallelized microslit isolator for physical property-based capture of tumor cells.

Numerous attempts have been made to develop efficient systems to purify trace amounts of circulating tumor cells (CTCs) from blood samples. However, current technologies are limited by complexities in device fabrication, system design, and process operability. Here we describe a facile, scalable, and highly efficient approach to physically capturing CTCs using a rationally designed microfluidic isolator with an array of microslit channels. The wide but thin microslit channels with a depth of several micrometers selectively capture CTCs, which are larger and less deformable than other blood cells, while allowing other blood cells to just flow through. We investigated in detail the effects of the microchannel geometry and operating parameters on the capture efficiency and selectivity of several types of cultured tumor cells spiked in blood samples as the CTC model. Additionally, in situ post-capture staining of the captured cells was demonstrated to investigate the system's applicability to clinical cancer diagnosis. The presented approach is simple in operation but significantly effective in capturing specific cells and hence it may have great potential in implementating cell physics-based CTC isolation techniques for cancer liquid biopsy.

Formation of 3D tissues of primary hepatocytes using fibrillized collagen microparticles as intercellular binders.

Numerous attempts have been made to organize isolated primary hepatocytes into functional three-dimensional (3D) constructs, but technologies to introduce extracellular matrix (ECM) components into such assemblies have not been fully developed. Here we report a new approach to forming hepatocyte-based 3D tissues using fibrillized collagen microparticles (F-CMPs) as intercellular binders. We created thick tissues with a thickness of ∼200 µm simply by mixing F-CMPs with isolated primary rat hepatocytes and culturing them in cell culture inserts. Owing to the incorporated F-CMPs, the circular morphology of the formed tissues was stabilized, which was strong enough to be manually manipulated and retrieved from the chamber of the insert. We confirmed that the F-CMPs dramatically improved the cell viability and hepatocyte-specific functions such as albumin production and urea synthesis in the formed tissues. The presented approach provides a versatile strategy for hepatocyte-based tissue engineering, and will have a significant impact on biomedical applications and pharmaceutical research.

Polyanion-induced, microfluidic engineering of fragmented collagen microfibers for reconstituting extracellular environments of 3D hepatocyte culture.

Artificial biological scaffolds made of extracellular matrix (ECM) components, such as type I collagen, provide ideal physicochemical cues to various cell culture platforms. However, it remains a challenge to fabricate micrometer-sized ECM materials with precisely controlled morphologies that could reconstitute the 3-dimensional (3D) microenvironments surrounding cells. In the present study, we proposed a unique process to fabricate fragmented collagen microfibers using a microfluidic laminar-flow system. The continuous flow of an acidic collagen solution was neutralized to generate solid fibers, which were subsequently fragmented by applying a gentle shear stress in a polyanion-containing phosphate buffer. The morphology of the fiber fragment was controllable in a wide range by changing the type and/or concentration of the polyanion and by tuning the applied shear stress. The biological benefits of the fragmented fibers were investigated through the formation of multicellular spheroids composed of primary rat hepatocytes and microfibers on non-cell-adhesive micro-vessels. The microfibers enhanced the survival and functions of the hepatocytes and reproduced proper cell polarity, because the fibers facilitated the formation of cell-cell and cell-matrix interactions while modulating the close packing of cells. These results clearly indicated that the microengineered fragmented collagen fibers have great potential to reconstitute extracellular microenvironments for hepatocytes in 3D culture, which will be of significant benefit for cell-based drug testing and bottom-up tissue engineering.

Sacrificial Alginate-Assisted Microfluidic Engineering of Cell-Supportive Protein Microfibers for Hydrogel-Based Cell Encapsulation.

Although many types of technologies for hydrogel-based cell cultivation have recently been developed, strategies to integrate cell-adhesive micrometer-sized supports with bulk-scale hydrogel platforms have not been fully established. Here, we present a highly unique approach to produce cell-adhesive, protein-based microfibers assisted by the sacrificial template of alginate; we applied these fibers as microengineered scaffolds for hydrogel-based cell encapsulation. Two types of microfluidic devices were designed and fabricated: a single-layered device for producing relatively thick (Φ of 10-60 µm) alginate-protein composite fibers with a uniform cross-sectional morphology and a four-layered device for preparing thinner (Φ of ∼4 µm) ones through the formation of patterned microfibers with eight distinct alginate-protein composite regions. Following chemical cross-linking of protein molecules and the subsequent removal of the sacrificial alginate from the double-network matrices, microfibers composed only of cross-linked proteins were obtained. We used gelatin, albumin, and hemoglobin as the protein material, and the gelatin-based cell-adhesive fibers were further encapsulated in hydrogels together with the mammalian cells. We clarified that the thinner fibers were especially effective in promoting cell proliferation, and the shape of the constructs was maintained even after removing the hydrogel matrices. The presented approach offers cells with biocompatible solid supports that enhance cell adhesion and proliferation, paving the way for the next generation of techniques for tissue engineering and multicellular organoid formation.

Laborless, Automated Microfluidic Tandem Cell Processor for Visualizing Intracellular Molecules of Mammalian Cells.

Visualization and quantification of intracellular molecules of mammalian cells are crucial steps in clinical diagnosis, drug development, and basic biological research. However, conventional methods rely mostly on labor-intensive, centrifugation-based manual operations for exchanging the cell carrier medium and have limited reproducibility and recovery efficiency. Here we present a microfluidic cell processor that can perform four-step exchange of carrier medium, simply by introducing a cell suspension and fluid reagents into the device. The reaction time period for each reaction step, including fixation, membrane permeabilization, and staining, was tunable in the range of 2 to 15 min by adjusting the volume of the reaction tube connecting the neighboring exchanger modules. We double-stained the cell nucleus and cytoskeleton (F-actin) using the presented device with an overall reaction period of ∼30 min, achieving a high recovery ratio and high staining efficiency. Additionally, intracellular cytokine (IL-2) was visualized for T cells to demonstrate the feasibility of the device as a pretreatment system for downstream flow-cytometric analysis. The presented approach would facilitate the development of laborless, automated microfluidic systems that integrate cell processing and analysis operations and would pave a new path to high-throughput biological experiments.

Enhanced Immunoadsorption on Imprinted Polymeric Microstructures with Nanoengineered Surface Topography for Lateral Flow Immunoassay Systems.

Lateral flow immunoassay devices have revolutionized the style of on-site disease detection and point-of-care testing in the past few decades. The surface nanotopography of a solid substrate is a dominant parameter in the efficiency of antibody immobilization, but precise control over surface roughness has not been fully investigated. Here we presented lateral flow immunoassay platforms with nanometer-scale surface roughness, reproducibly engineered using thermal nanoimprinting lithography, and investigated the effects of surface nanotopography on immunoadsorption and immunoassay performance. We fabricated three types of imprinted polycarbonate sheets with microcone array structures having different degrees of surface roughness using three types of molds fabricated by micromachining or laser ablation. The structures fabricated by laser-ablated nickel mold exhibited numerous bumps measuring several tens of nanometers, which enhanced antibody adsorption. We performed sandwich immunoassays of C-reactive protein in serum samples and achieved highly sensitive detection with a detection limit of ∼0.01 µg mL-1 and a broad dynamic range. The present results provide useful information on the remarkable effect of nanoengineered surfaces on biomolecule adsorption, and the platforms presented here will widen the applicability and versatility of lateral flow immunoassay devices.

Hydrodynamic Microparticle Separation Mechanism Using Three-Dimensional Flow Profiles in Dual-Depth and Asymmetric Lattice-Shaped Microchannel Networks.

We herein propose a new hydrodynamic mechanism of particle separation using dual-depth, lattice-patterned asymmetric microchannel networks. This mechanism utilizes three-dimensional (3D) laminar flow profiles formed at intersections of lattice channels. Large particles, primarily flowing near the bottom surface, frequently enter the shallower channels (separation channels), whereas smaller particles flowing near the microchannel ceiling primarily flow along the deeper channels (main channels). Consequently, size-based continuous particle separation was achieved in the lateral direction in the lattice area. We confirmed that the depth of the main channel was a critical factor dominating the particle separation efficiencies, and the combination of 15-µm-deep separation channels and 40-µm-deep main channels demonstrated the good separation ability for 3-10-µm particles. We prepared several types of microchannels and successfully tuned the particle separation size. Furthermore, the input position of the particle suspension was controlled by adjusting the input flow rates and/or using a Y-shaped inlet connector that resulted in a significant improvement in the separation precision. The presented concept is a good example of a new type of microfluidic particle separation mechanism using 3D flows and may potentially be applicable to the sorting of various types of micrometer-sized objects, including living cells and synthetic microparticles.

A numbering-up strategy of hydrodynamic microfluidic filters for continuous-flow high-throughput cell sorting.

Even though a number of microfluidic systems for particle/cell sorting have been proposed, facile and versatile platforms that provide sufficient sorting throughput and good operability are still under development. Here we present a simple but effective numbering-up strategy to dramatically increase the throughput of a continuous-flow particle/cell sorting scheme based on hydrodynamic filtration (HDF). A microfluidic channel equipped with multiple branches has been employed as a unit structure for size-based filtration, which realizes precise sorting without necessitating sheath flows. According to the concept of resistive circuit models, we designed and fabricated microdevices incorporating 64 or 128 closely assembled, multiplied units with a separation size of 5.0/7.0 µm. In proof-of-concept experiments, we successfully separated single micrometer-sized model particles and directly separated blood cells (erythrocytes and leukocytes) from blood samples. Additionally, we further increased the unit numbers by laminating multiple layers at a processing speed of up to 15 mL min-1. The presented numbering-up strategy would provide a valuable insight that is universally applicable to general microfluidic particle/cell sorters and may facilitate the actual use of microfluidic systems in biological studies and clinical diagnosis.

One-Step Formation of Microporous Hydrogel Sponges Encapsulating Living Cells by Utilizing Bicontinuous Dispersion of Aqueous Polymer Solutions.

With the recent progress in three-dimensional (3D) cell culture techniques for regenerative medicine and drug development, hydrogel-based tissue engineering approaches that can precisely organize cells into functional formats have attracted increasing attention. However, challenges remain in creating continuous microconduits within hydrogels to effectively deliver oxygen and nutrients to the embedded cells. Here we propose a one-step, fully liquid state, and all-aqueous process to create porous hydrogels that can encapsulate living cells without the need for extensive processing protocols, including the incorporation and removal of sacrificial materials. An unusual bicontinuous state of aqueous two-phase dispersion was utilized, and one of the two phases, encapsulating living cells, was rapidly photo-cross-linked to form hydrogel sponges. We optimized the volumetric mixing ratio of gelatin methacrylate (GelMA)-rich and polyethylene glycol (PEG)-rich solutions and investigated the effects of the formed continuous microconduits on the cell functions by creating liver-tissue mimetic 3D constructs. The presented technology provides a facile and versatile strategy for fabricating microstructured hydrogels for cell culture and would bring new insights for the development of porous materials by fully aqueous bicontinuous dispersions.

Formation of pressurizable hydrogel-based vascular tissue models by selective gelation in composite PDMS channels.

Vascular tissue models created in vitro are of great utility in the biomedical research field, but versatile, facile strategies are still under development. In this study, we proposed a new approach to prepare vascular tissue models in PDMS-based composite channel structures embedded with barium salt powders. When a cell-containing hydrogel precursor solution was continuously pumped in the channel, the precursor solution in the vicinity of the channel wall was selectively gelled because of the barium ions as the gelation agent supplied to the flow. Based on this concept, we were able to prepare vascular tissue models, with diameters of 1-2 mm and with tunable morphologies, composed of smooth muscle cells in the hydrogel matrix and endothelial cells on the lumen. Perfusion culture was successfully performed under a pressurized condition of ∼120 mmHg. The presented platform is potentially useful for creating vascular tissue models that reproduce the physical and morphological characteristics similar to those of vascular tissues in vivo.

Thermally imprinted microcone structure-assisted lateral-flow immunoassay platforms for detecting disease marker proteins.

Although various types of on-site immunoassay platforms have been developed, facile and reliable sample-to-answer immunoassay systems are still under development. In this study, we proposed a lateral-flow immunoassay system utilizing a polymer sheet with microcone array structures fabricated by thermal imprinting. By adding a surfactant to the running/washing buffer, we were able to dispense with complicated chemical modification protocols, which are usually necessary to enhance antibody adsorption. We investigated three types of polymeric materials and confirmed that polycarbonate is most suitable as an imprinted polymer substrate. Experiments using microcone arrays with different distances revealed that the increased surface area with nanometer-scale surface roughness was key to achieving stable immobilization of antibodies. To assess the applicability of this assay to clinical diagnosis, C-reactive protein in a pure buffer and in a serum sample was analyzed as a model, with a ∼0.1 µg mL-1 lower limit of detection. The presented approach would open a new path to the development of immunoassay-based on-site point-of-care testing by virtue of its simplicity of operation, high sensitivity, and versatility.

Micropassage-embedding composite hydrogel fibers enable quantitative evaluation of cancer cell invasion under 3D coculture conditions.

Cell migration and invasion are of significant importance in physiological phenomena, including wound healing and cancer metastasis. Here we propose a new system for quantitatively evaluating cancer cell invasion in a three-dimensional (3D), in vivo tissue-like environment. This system uses composite hydrogel microfibers whose cross section has a relatively soft micropassage region and that were prepared using a multilayered microfluidic device; cancer cells are encapsulated in the core and fibroblasts are seeded in the shell regions surrounding the core. Cancer cell proliferation is guided through the micropassage because of the physical restriction imposed by the surrounding solid shell regions. Quantitative analysis of cancer cell invasion is possible simply by counting the cancer cell colonies that form outside the fiber. This platform enables the evaluation of anticancer drug efficacy (cisplatin, paclitaxel, and 5-fluorouracil) based on the degree of invasion and the gene expression of cancer cells (A549 cells) with or without the presence of fibroblasts (NIH-3T3 cells). The presented hydrogel fiber-based migration assays could be useful for studying cell behaviors under 3D coculture conditions and for drug screening and evaluation.

Development of a perfusable 3D liver cell cultivation system via bundling-up assembly of cell-laden microfibers.

Although the reconstruction of functional 3D liver tissue models in vitro presents numerous challenges, it is in great demand for drug development, regenerative medicine, and physiological studies. Here we propose a new approach to perform perfusion cultivation of liver cells by assembling cell-laden hydrogel microfibers. HepG2 cells were densely packed into the core of sandwich-type anisotropic microfibers, which were produced using microfluidic devices. The obtained microfibers were bundled up and packed into a perfusion chamber, and perfusion cultivation was performed. We evaluated cell viability and functions, and also monitored the oxygen consumption. Furthermore, fibers covered with vascular endothelial cells were united during the perfusion culture, to form vascular network-like conduits between fibers. The presented technique can structurally mimic the hepatic lobule in vivo and could prove to be a useful model for various biomedical research applications.

Direct Observation of Splitting in Oil-In-Water-In-Oil Emulsion Droplets via a Microchannel Mimicking Membrane Pores.

Direct observation of double emulsion droplet permeation through a microchannel that mimicked 100 µm membrane pores with a porosity of 66.7% provided insights regarding splitting mechanisms in porous membranes. The microchannel was fabricated by standard soft lithography, and the oil-in-water-in-oil double emulsion droplets were prepared with a glass capillary device. By changing the flow rate from 0.5 to 5.0 × 10-2 m s-1, three characteristic behaviors were observed: (a) passage into one channel without splitting; (b) division into two smaller components; and (c) stripping of the middle water phase of the double emulsion droplets into a smaller double emulsion droplet and a smaller water-in-oil single emulsion droplet. The mechanisms are discussed with respect to the balance of viscous forces and interfacial tension, the contact point with the tip of the channel, and the relative position of the innermost droplet within the middle droplet.

Collagen Microparticle-Mediated 3D Cell Organization: A Facile Route to Bottom-up Engineering of Thick and Porous Tissues.

In closely packed artificial 3D cellular constructs, cells located near the center of the constructs are not functional because of the limited supply of oxygen and nutrition. Here we describe a simple, unique, and highly versatile approach to organizing cells into thick but porous 3D tissues, using cell-sized collagen microparticles as particulate scaffolds. When cells and particles are mixed and seeded in a noncell-adhesive planar chamber, they gather to form sheet-shaped structures with a thickness of 100-150 µm. In the construct, uniformly distributed particles work as a binder between cells and modulate the strong intercellular contraction. We confirmed that several factors, including the particle/cell ratio and particle size, critically affect the stability and shrink behaviors of porous tissues prepared using mouse embryonic fibroblasts (NIH-3T3 cells). Cross-sectional observation, together with cell proliferation and viability assays, revealed that the cells composing the tissues are functional primarily because interior pores between cells/particles worked as a path for efficient molecular transport. Furthermore, we prepared thick cell tissues of a liver model using human hepatocarcinoma cells (HepG2 cells), and confirmed that liver-specific functions were upregulated when composite tissues were formed using collagen microparticles prepared with several different stabilization protocols by glutaraldehyde, genipin, and methyl acetate). The process presented would be highly useful in enabling one-step production of thick cellular constructs in which porosity and morphology are tunable.

Slanted, asymmetric microfluidic lattices as size-selective sieves for continuous particle/cell sorting.

Hydrodynamic microfluidic platforms have been proven to be useful and versatile for precisely sorting particles/cells based on their physicochemical properties. In this study, we demonstrate that a simple lattice-shaped microfluidic pattern can work as a virtual sieve for size-dependent continuous particle sorting. The lattice is composed of two types of microchannels ("main channels" and "separation channels"). These channels cross each other in a perpendicular fashion, and are slanted against the macroscopic flow direction. The difference in the densities of these channels generates an asymmetric flow distribution at each intersection. Smaller particles flow along the streamline, whereas larger particles are filtered and gradually separated from the stream, resulting in continuous particle sorting. We successfully sorted microparticles based on size with high accuracy, and clearly showed that geometric parameters, including the channel density and the slant angle, critically affect the sorting behaviors of particles. Leukocyte sorting and monocyte purification directly from diluted blood samples have been demonstrated as biomedical applications. The presented system for particle/cell sorting would become a simple but versatile unit operation in microfluidic apparatus for chemical/biological experiments and manipulations.

Fabrication of multilayered vascular tissues using microfluidic agarose hydrogel platforms.

Vascular tissues fabricated in vitro are useful tools for studying blood vessel-related cellular physiologies and for constructing relatively large 3D tissues. An efficient strategy for fabricating vascular tissue models with multilayered, branched, and thick structures through the in situ hydrogel formation in fluidic channels is proposed. First, an aqueous solution of RGD-alginate containing smooth muscle cells (SMCs) is introduced into channel structures made of agarose hydrogel, forming a cell-embedding Ca-alginate hydrogel layer with a thickness of several hundred micrometers on the channel surface because of the Ca2+ ions diffused from the agarose hydrogel matrix. Next, endothelial cells (ECs) are introduced and cultured for up to seven days to form hierarchically organized, multilayered vascular tissues. The factors affecting the thickness of the Ca-alginate hydrogel layer, and prepared several types of microchannels with different morphologies are examined. The fabricated vascular tissue models are easily recovered from the channel by simply detaching the agarose hydrogel plates. In addition, the effect of O2 tension (20 or 80%) on the viability and elastin production of SMCs during the perfusion culture is evaluated. This technique would pave a new way for vascular tissue engineering because it enables the facile production of morphologically in vivo vascular tissue-like structures that can be employed for various biomedical applications.

Microfluidic System Enabling Multistep Tuning of Extraction Time Periods for Kinetic Analysis of Droplet-Based Liquid-Liquid Extraction.

When analyzing the kinetics of liquid-liquid extraction (LLE), the change in the concentration of extracted target molecules over time should be monitored for a known interfacial area. Herein, we developed a microfluidic system for precisely analyzing the kinetics of LLE using droplets of a constant size even in the presence of additives. Extraction is initiated by exchanging the carrier fluid for the droplets with a target solution and then terminated by phase separation, based on the principle of hydrodynamic filtration. By using one out of several pairs of outlet/buffer inlet at a given time, the extraction time period is tuned stepwise without changing the flow rate condition. We successfully demonstrated droplet-based LLE by controlling the extraction period from ∼0.03 to ∼1.2 s and evaluated the extraction kinetics of rhodamine B from the continuous aqueous phase to droplets of 1-octanol with a diameter of ∼40 µm. In addition, the effect of additives on extraction efficiency was evaluated. The system presented in this study would be useful for determining rate constants for interfacial mass transfer in general LLE kinetic studies as well as for developing new extraction chemistries and optimizing microfluidic chemical/biochemical analysis systems that involve an LLE process.